Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
  • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
  • A61K31/404—Indoles, e.g. pindolol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/415—1,2-Diazoles
  • A61K31/4155—1,2-Diazoles non condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0048—Eye, e.g. artificial tears
  • A61K9/0051—Ocular inserts, ocular implants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/06—Antiglaucoma agents or miotics

Definitions

• AMD Age related macular degeneration
• AMD Age related macular degeneration
• atrophic or dry AMD neovascular or wet AMD.
• AMD begins as dry AMD.
• Dry AMD is characterized by the formation of yellow plaque like deposits called drusen in the macula, between the retinal pigment epithelium (RPE) and the underlying choroid.
• RPE retinal pigment epithelium
• 5-lipoxygenase (5-lipoxygenase, 5-LO or Alox5) is a member of the lipoxygenase family of enzymes and increases with aging.
• 5-LO converts arachidonic acid into the powerful inflammatory leukotrienes. Leukotrienes promote cancer, damage the brain, promote asthma, arthritis, psoriasis and ulcerative colitis (Steele VE et al.
• Lipoxygenase inhibitors as potential cancer chemopreventives, Cancer Epidemiol Biomarkers Prev 1999, 8:467-83). They may also promote atherosclerosis (Rasmark O. Editorial: 5-lipoxygenase-derived leukotrienes. Mediators also of atherosclerotic inflammation. Arterioscler Thromb Vase Biol 2003, 23: 1140-42. 57. Kiecolt-Glaser JK, et al. Chronic stress and age-related increases in the proinflammatory cytokine IL-6.
• the 5 -lipoxygenase enzyme is activated by glucocorticoids (Cortisol), (Manev H et al. Putative role of neuronal 5-lipoxygenase in an aging brain. FASEB J 2000, 14: 1464-69) and inhibited by melatonin.
• Cortisol glucocorticoids
• melatonin-6 increase Cortisol in humans, (Dean W. Adaptive homeostat dysfunction. Vit Res News 2005, 19(2): 1-5), while melatonin decreases drastically with aging, (Dean W. Neuroendocrine theory of aging: an introduction. Vit Res News 2005, 19(1): 1-4.)
• This invention provides a method of treating age related macular degeneration and ocular ischemic disease associated with complement activation by targeting 5- lipoxygenase.
• Figure 1 shows that Anti-ARPE-19 serum leads to complement activation.
• S- 58 Solid line
• S-60 dotted line
• polyclonal serum recognize ARPE-19 cells as determined by FACS analysis.
• S-58 Induces complement activation as determined by generation of C3a
• C Soluble C5b-9
• D S-58 induction of C5b-9 deposition on surface of ARPE19 cells as determined by FACS
• D S-58 (Solid line) S-60 (Dotted line)
• Dose dependent cell death and ATP release C
• a functional alternative complement cascade is required for S-58 induced complement mediated cell death (D).
• Figure 3 shows that oxidative stress induced by H 2 0 2 or t-BH leads to enhanced cell death(A) although this increase in cell death does not correlate with altered expression of cRegs on the cell surface (B).
• Figure 4 shows a scatter plot of results from library screen.
• Figure 5 shows examples of compounds with protective properties.
• Figure 6 shows that the 5 -lipoxygenase activating protein (FLAP) inhibitor MK886 protects ARPE-19 cells from oxidative stress and complement attack
• FLAP 5 -lipoxygenase activating protein
• Figure 7 shows dose dependent protection of ARPE-19 cells against cell death by the lipoxygenase inhibitor 2-TEDC.
• Figure 8 shows The 15 lipoxygenase inhibitor PD 146176 has no protective activity in blocking cell death mediated by the synergy between complement attach and oxidative stress.
• Figure 9 shows that minocyclin dose dependently protects ARPE-19 cells against cell death mediated by the synergy between complement attack and oxidative stress.
• Figure 10 shows that the 5 -lipoxygenase inhibitor PF-4191834 protected against functional loss of retinal ERG A and B-wave amplitudes (Figs. 10 A and B, respectively) in animals in response to oxidative stress induced by intravitreal PQ injection. (* p ⁇ 0.05 in student tTest)
• Figure 11 shows that the 5 -lipoxygenase inhibitor PF-4191834 protected against functional loss of retinal ERG A- wave amplitudes in animals in response to blue light induced retinal damage. (* p ⁇ 0.05 in student tTest)
• Figure 12 shows the scoring template used for evaluation of retinal damage in animals in response to light induced damage.
• Figure 13 shows a comparison of retinal structure and outer nuclear layer thickness in vehicle and 5-lox inhibitor treated animals after blue light induced retinal damage. Note, that in animals receiving 5-lox inhibitor have a higher level of protection in ONL thickness relative to vehicle treated animals.
• the subject in need can be a human or non-human animal, including a mouse, rat, rabbit, or non-human primate, or any other non-human mammal suffering from or in need of treatment of AMD or ocular ischemic disease.
• One embodiment of the present invention is a method for treating AMD or ocular ischemic disease in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a compound that reduces 5 -lipoxygenase (5-LO) activity.
• a compound that reduces 5 -lipoxygenase (5-LO) activity is administered to said subject a therapeutically effective amount of a compound that reduces 5 -lipoxygenase (5-LO) activity.
• Another embodiment of the present invention is a method for treating AMD or ocular ischemic disease in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a compound that reduces 5 -lipoxygenase (5- LO) activity by inhibiting 5 -lipoxygenase activating protein (FLAP).
• the compound that reduces 5 -lipoxygenase activity is one that targets the 5 -lipoxygenase polypeptide or a gene encoding the 5-LO polypeptide, thereby inhibiting or reducing the activity of the 5- LO polypeptide.
• the compound is one that selectively inhibits 5-lipoxygenase.
• examples of compounds that inhibit the expression of a polypeptide, and thereby the activity of a polypeptide include siRNAs (short interfering RNAs, double stranded RNAs of 21-23 nucleotides in length), antisense nucleic acids, and ribozymes.
• the compound that selectively inhibits 5 -lipoxygenase is PF- 4191834 (Masferrer et al. (2010) J. Pharm, Experimental Therapeutics 334(1):294-301).
• the compound that directly or selectively inhibits 5 -lipoxygenase and reduces 5-lipoxgenase activity is Zileuton.
• the method comprises administering a compound that inhibits or reduces 5 -lipoxygenase activity by inhibiting 5 -lipoxygenase activating protein (FLAP) in an amount effective for treating AMD or ocular ischemic disease.
• FLAP 5 -lipoxygenase activating protein
• the compound that selectively inhibits FLAP may be a small molecule, an antibody, an aptamer, an antisense nucleic acid, or a small interfering RNA (siRNA).
• the compound that inhibits FLAP is MK886.
• Compounds for use in the present methods of treating age-related macular degeneration (AMD) and ocular ischemic disease can be any substance that selectively inhibits or reduces the expression, function, or activity of 5 -lipoxygenase (5-LO) in the eye of an individual.
• ALD age-related macular degeneration
• ocular ischemic disease can be any substance that selectively inhibits or reduces the expression, function, or activity of 5 -lipoxygenase (5-LO) in the eye of an individual.
• Exemplary compounds that may be used to inhibit the expression or activity of 5 -lipoxygenase include short interfering double stranded RNAs (siRNAs) directed to the gene encoding 5 -lipoxygenase, single stranded antisense nucleic acids targeted to the gene encoding 5 -lipoxygenase, or ribozymes targeted to the gene encoding 5 -lipoxygenase, each of which can be used to reduce the expression of 5-LO in the eye of an individual in need.
• siRNAs short interfering double stranded RNAs
• the practitioner can use monoclonal or polyclonal antibodies or aptamers targeted to 5-LO, or small molecule compounds that selectively or directly inhibit the activity of the 5-LO enzyme in the eye of an individual in need.
• the practitioner can selectively reduce 5-LO activity by administering a compound that inhibits 5 -lipoxygenase activating protein (FLAP).
• FLAP 5 -lipoxygenase activating protein
• Short interfering RNAs sometimes referred to as small interfering RNAs, or RNAi compounds, are designed to hybridize to the nucleotide sequence of the mRNA target.
• Exemplary nucleotide sequences of mRNAs encoding human 5-LO and FLAP are known, as shown by Table 1, below.
• the nucleotide and polypeptide sequences defined by the GenBank Accession numbers given below in Table 1 are hereby incorporated by reference. See also Table 1 in U.S. Patent 7,829,535, which is hereby incorporated by reference, describing 5-LO and FLAP mRNAs from other mammalian species such as Rat and Mouse.
• Table 1 GenBank Accession Numbers of exemplary m NA and Polypeptide Sequences for arachidonate 5 -lipoxygenase, also known as 5 -lipoxygenase, or 5-LO.
• the compound for inhibiting 5 -lipoxygenase can have the formula:
• Ri is hydrogen (H) or fluorine (F).
• PF-4191834 is a selective non-redox 5 -lipoxygenase inhibitor and is described in Masferrer, J.L., et al., Pharmacology of PF-4191834), a selective non- redox 5 -lipoxygenase inhibitor. Effective in inflammation and pain. J Pharmacol Exp Ther. 334(1): p. 294-301
• the small molecule compound for reducing 5- lipoxygenase activity can be any of those listed in Figure 5 of this application.
• the small molecule compound for selectively inhibiting 5 -lipoxygenase in the methods described herein can be racemic Zileuton (ZYFLO®), known as ⁇ -[l-(l- benzothien-2-yl)ethyl]-N-hydroxyurea.
• Zileuton is a hydroxyl urea having a
• an enantiomerically pure form of Zileuton such as (R)-Zileuton
• (R)-Zileuton may be useful in the present method to inhibit 5-lipoxygenase.
• the chemical structure of (R)-Zileuton is described in U.S. Patent Application Publication 2010/0273868, which is hereby incorporated by reference. See in particular pages 1-3 of U.S. 2010/0273868, describing the chemical structure of Zileuton and methods of synthesizing racemic and (R)-Zileuton.
• the Zileuton used in the present methods may have the formula defined by any of claims 1-12 of U.S. Patent 4,873,259, which is hereby incorporated by reference.
• the small molecule compound can be (2S,4R)-4-(2-fluoro-3- ⁇ [4-( 1 -methyl- 1 H-pyrazol-5 -yl)phenyl]thio ⁇ phenyl)-2-methyltetrahydro-2H-pyran-4- carbonitrile having the formula:
• Hofmann et al. ((2011) J. Med. Chem. 54, 1943-1947) teaches that 5-benzylidene-2-phenylthiazolinones act as potent direct 5-LO inhibitors and teaches a core structure, or thiazolinone scaffold, common to this class of inhibitors.
• 5-LO inhibitors having the core formula:
• Hofmann et al. show a class of 5-LO inhibitors having the formula:
• Patent 7,829,535 teaches that a 5-lipoxygenase inhibitor can be 3 - [ 1 -(4-chlorobenzyl)-3 -t-butyl-thio-5 - isopropylindol-2-yl] -2,2-dimethylpropanoic acid (MK886); 3-(l-(4-chlorobenzyl)-3-(l- butyl-thio)-5-(quinolin-2-yl-methoxy)-indol-2-yl)-2,2-dimethyl propanoic acid) (MK-591); nordihydroguaiaretic acid (NDGA); 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl- 1 ,4-benzoquinone (AA861); or (N-(l-benzo(b)thien-2-ylethyl)-N-hydroxyurea) (Zileuton).
• MK886 3-(l
• U.S. Patent 7,829,535 further teaches that 5-lipoxygenase inhibitors include tenidap, zileuton, flobufen, lonapalene, tagorizine, Abbott A-121798, Abbott A-76745, Abbott A- 78773, Abbott A-79175, Abbott ABT 761, Ciba-Geigy CGS-26529, Biofor BF-389, Cytomed CMI-392, Leo Denmark ETH-615, lonapalene, Merck Frosst L 699333, Merckle ML-3000, 3M Pharmaceuticals R-840, linazolast (TMK-688), Zeneca ZD-7717, Zeneca ZM-216800, Zeneca ZM-230487, and Zeneca ZD-2138.
• 5-lipoxygenase inhibitors include tenidap, zileuton, flobufen, lonapalene, tagorizine, Abbott A-121798, Abbott A-76745, Abbott A
• U.S. Patent 6,455,541 which is hereby incorporated by reference, teaches the small molecule 5-LO inhibitors NGDA, MK886, and ZM230,487. See in particular Figs. 9-11 in U.S. Patent 6,455,541, hereby incorporated by reference.
• U.S. Patent 4,634,766 which is hereby incorporated by reference, teaches a class of small molecule 1 ,4-diaza-phenothiazines that are said to be inhibitors of mammalian 5 -lipoxygenase. See in particular the compounds defined by claims 1-8 in U.S. Patent 4,634,766, hereby incorporated by reference.
• the compounds When used in combination, the compounds may be administered together or separately at intervals, with or without resting periods between administrations.
• ALD age related macular degeneration
• compounds that inhibit 5- lipoxygenase may be used to treat atrophic or dry AMD and neo vascular or wet AMD.
• the present invention is also directed to the preparation of a medicament, or pharmaceutical composition, for the treatment AMD.
• the medicament contains a pharmaceutical acceptable composition, which comprises a therapeutically effective amount of a compound that inhibits 5-LO, as described herein, together with a pharmaceutical acceptable carrier.
• the medicament or pharmaceutical composition may further contain a second or additional therapeutic agent for the treatment of AMD.
• compositions can be used as a medicament and
• 5-LO inhibitory compounds of the present invention are well known in the art, and can be readily adapted for delivery of 5-LO inhibitory compounds of the present invention.
• Methods for administering 5-LO inhibitory compounds and compositions thereof to the subject in need of treatment of AMD include, but are not limited to, oral, systematic, parenteral, local, and topical delivery.
• the dosage forms can be tablets, capsules, intravenous injections, intramuscular injections, local injections, topical creams, gels and ointments, eye drops, ophthalmic solutions, ophthalmic suspensions, ophthalmic emulsions, intravitreal injections, subtenon injections, ophthalmic bioerodible implant, and non-bioerodible ophthalmic inserts or depots, nasal sprays and ointment, various rectal or vaginal preparations.
• compounds and pharmaceutical compositions that selectively or indirectly inhibit 5-LO can be administered systemically, orally, subcutaneously, intravenously, intraperitoneal, intravitreal, retrobulbar, with a bierodible implant drug delivery system into the eye of a subject, or topically to the eye, or by injecting a biodegradable drug delivery system containing a 5-LO inhibitor into the eye of the subject such as the vitreous cavity of the eye.
• Bioerodible or biodegradable ocular implants and biodegradable (e.g., PLA and/or PLGA-containing) polymer drug delivery systems that may be useful for administration of a 5 -lipoxygenase inhibitory compound in the present methods are described in U.S. Patent Application Publication 2010/0015158, which is hereby incorporated by reference. See in particular pages 7-9 (paragraphs 71-94) in US
• one embodiment of the present invention is an intraocular biodegradable drug delivery system comprising a 5 -lipoxygenase inhibitor (i.e., a compound that reduces 5 -lipoxygenase activity) and a biodegradable polymer such as a poly(lactide) (PLA) polymer or a poly(lactide-co-glycolide) (PLGA) polymer or a combination of PLA and PLGA polymers.
• the drug delivery system can be injected or implanted into an intraocular location to provide sustained release of the 5-LO inhibitor.
• Polylactide (PLA) polymers exist in two chemical forms, poly(L-lactide) and poly(D,L-lactide).
• a PLGA is a co- polymer that combines poly(D,L-lactide) with poly(glycolide) in various possible ratios. The higher the glycolide content in a PLGA the faster the polymer degradation.
• a drug delivery system for intraocular administration (i.e. by intravitreal implantation or injection) of a 5-LO inhibitor comprises, consists of, or consists essentially of a 5-LO inhibitor and at least a 75 weight percent of a PLA and no more than about a 25 weight percent of a poly(D,L-lactide -co-glycolide) polymer.
• suspensions of microspheres incorporating a 5-LO inhibitor suspended in a hydrogel such as a polymeric hyaluronic acid
• a hydrogel such as a polymeric hyaluronic acid
• Another embodiment of the present invention includes using a
• biodegradable drug delivery system comprising a 5-LO inhibitor in the eye of a subject (e.g., a human individual or non-human mammal or other animal) suffering from AMD or related ocular condition to relieve, reduce, or protect against one or more symptoms associated with age related macular degeneration (AMD) or related ocular condition.
• a subject e.g., a human individual or non-human mammal or other animal
• AMD age related macular degeneration
• One embodiment of the present invention is a method comprising placing a biodegradable drug delivery system comprising a 5-LO inhibitor and a biodegradable polymer or polymer mixture (e.g., PLA or PLGA or both) in the eye of an individual suffering from AMD or at risk of developing AMD.
• the individual may be a human or non-human animal, including but not limited to a rabbit, mouse, horse, dog, rat, or monkey.
• the 5-LO inhibitory compound may be combined and delivered together with a second therapeutic agent for the treatment of AMD.
• ASD Age-related macular degeneration
• small molecule compounds and “small molecules” do not include polypeptides or polynucleotides and are preferably less than about 1000 kDa.
• Therapeutic levels means an amount or a concentration of an active agent that has been locally delivered to an ocular region that is appropriate to treat an ocular condition such as AMD so as to reduce or prevent a symptom of an ocular condition such as AMD.
• Treating means to administer a treatment to a human or non-human subject to obtain a therapeutic effect. Treating may include reducing or slowing a clinical symptom associated with an ocular condition such as AMD, including but not limited to vision loss and/or neovascularization.
• Oxidative stress and complement activation are the two most highly cited factors associated with occurrence and progression of AMD.
• To examine functional consequences of this relationship we treated ARPE-19 cells with either H 2 0 2 or t-BH followed by challenge with complement.
• Oxidative stress induced by t-BH caused a 20-30% increase in cell death while that of H 2 0 2 resulted in a >10 fold increase (Figure 3 A).
• ARPE-19 cells were plated at sub confluence in 96 well culture plates and cultured 24 to 48 hrs. The cells were then treated with 1 : 1000 dilution of each compound from the NIH clinical collection library overnight. The following day culture media was collected and set aside. The cells were washed and then treated with 0.77 mM H2O2 in hanks buffered saline solution (HBSS) for 90 minutes in the presence of fresh compound from NIH clinical collection library at a 1 : 1000 dilution . Cells were then washed and primed with a solution of HBSS containing 24% S-58 for 30 minutes.
• HBSS hanks buffered saline solution
• Figure 4 is the distribution of results indexed to compound plate (y-axis) and Protection (x-axis). Based on the distribution a break point at 60% and greater protection was chosen as a cutoff for compounds that showed significant protection in the assay (Boxed region in Figure 4).
• Figure 5 contains a list of compounds with protective properties. Of the 17 compounds selected, six compounds (Zeranol, Penciclovir, Rifapentine, Rifaximin, calcitrol, 3-[3,5-DIBROMO-4- HYDROXYBENZOYLJ-2-ETHYLBENZOFURAN) were deemed to function as antioxidants in the assay.
• the results identify modulators of 5 -lipoxygenase as therapeutic targets for AMD, including agents increasing cAMP kinase activity such as phospholiesterase inhibitors type IV, Prostaglandin E2, adenosine a2 receptor agonist, compounds inhibiting 5 -lipoxygenase activation protein (FLAP) as represented by MK- 0591 and MK-886, compounds modulating PLA2 activation, as well as compounds targeting p38 pathway and Rac activation.
• agents increasing cAMP kinase activity such as phospholiesterase inhibitors type IV, Prostaglandin E2, adenosine a2 receptor agonist
• FLAP 5 -lipoxygenase activation protein
• the 5 -lipoxygenase inhibitor MK886 protects ARPE-19 cells from oxidative stress and complement attack
• 5-LO-activating protein FLAP
• FLAP 5-LO-activating protein
• MK886 (l-[(4-chlorophenyl)methyl]-3-[(l,l-dimethylethyl)thio]-a,a- dimethyl-5-(l-methylethyl)-lH-indole-2-propanoic acid, sodium salt) has the formula:
• ARPE-19 cells from oxidative stress and complement attack in vitro The cell protection assay was carried out essentially as described in Examples 1-4.
• I.C.50 of 0.1 which correlates with the known I.C.50 (0.09 um) inhibition of 5- lipoxygenase by this compound (Cho, H., et al., Novel caffeic acid derivatives: extremely potent inhibitors of 12-lipoxygenase. J Med Chem, 1991. 34(4): p. 1503-5). This does not appear to involve either the 12 lipoxygenase or 15 lipoxygenase activity of the compound, since little activity is seen at the known I.C.
• 15 lipoxygenase inhibitor PD 146176 (6,1 l-dihydro-[l]benzothiopyrano[4,3-b]indol) (Bocan, T.M., et al, A specific 15 -lipoxygenase inhibitor limits the progression and monocyte-macrophage enrichment of hypercholesterolemia-induced atherosclerosis in the rabbit. Atherosclerosis, 1998. 136(2): p. 203-16).
• Minocyclin Song, Y., et al., "Minocycline protects PC12 cells from ischemic-like injury and inhibits 5-lipoxygenase activation" Neuroreport, 2004. 15(14): p. 2181-4.
• minocyclin like MK886 and 2-TEDC, dose dependently protected against complement and oxidative stress mediated cell death.
• 5-LO inhibitors protect against functional loss of retinal ERG A and B-wave amplitudes in vivo following oxidative stress induced by intravitreal PQ injection.
• the first in vivo model was a model of retinal oxidative stress induced by intravitreal injection of Paraquat in SOD 1-/+ animals (Dong, A., et al., Superoxide dismutase 1 protects retinal cells from oxidative damage. J Cell Physiol, 2006. 208(3): p. 516-26).
• Paraquat is also known to induce activation of complement (Sun, S., et al. (201 1 Mar.
• the 5-LO inhibitory compound used in these in vivo studies was PF-4191834, a selective non-redox 5 -lipoxygenase inhibitor. (Masferrer, J.L., et al, J Pharmacol Exp Ther. 334(1): pp. 294-301).
• PF-4191834 has the formula:
• mice received first and second oral doses of 1 mg/kg of PF- 4191834 sixteen (16) hrs and 4 hrs prior to the paraquat injection and then a third oral dose of 1 mg/kg of PF-4191834 four (4) hours after the paraquat injection.
• Control animals received equivalent volume of vehicle (0.5% Avicel R-91 and 0.1% Tween-80 in dH 2 0) to compound treated animals.
• a 1 ⁇ injection of 0.75 mM Paraquat was made using a Hamilton Lab animal injector (LASI 115) designed to deliver between 25 nanoliters to 25 microliters per injection. Injections were performed under a dissection microscope and a foot pedal to execute injections. Animals were revived by administration of 0.5 mg/kg atipamezole i.p., returned to their cages, and warmed using a heating pad and monitored until awake.
• LASI 115 Hamilton Lab animal injector
• the ERG was performed 1 day post paraquat injection. Scotopic ERGs were performed using a .001, .01, and 1 cd.s/m2 flash stimulus and 20 Hz flicker protocol. Mice were dark-adapted for a 12 hour period and ERG recordings were performed using the Espion ERG Diagnosys system. Pupils were first dilated with drops of both 1% AK- Pentolate (Cyclopentolate Hydrochloride) and 10% AK-Dilate (Phenylephrine
• mice were anesthetized with a ketamine (75 mg/kg) and dexmedetomidine (1.0 mg/kg) cocktail (injected i.p.). Celluvisc was placed on the eyes followed by surface electrodes. The ERG was then recorded with the animal on a heating pad (takes 7-8 minutes). Animals were revived by administration of 0.5 mg/kg atipamezole (injected i.p.), returned to their cages, and warmed using a heating pad and monitored until awake.
• atipamezole injected i.p.
• Balb/c mice were dosed orally (PO) at 1 mg/Kg starting 16 hours prior to blue light, and then BID for the next 14 days.
• Control animal received an equivalent volume of vehicle (0.5% Avicel R-91 and 0.1% Tween-80 in dH 2 0) to compound treated animals .
• the ERG was performed 2 weeks post blue light (4 days post OCT to allow for cataracts to clear). Scotopic ERGs were performed using a .001, .01, and 1 cd.s/m2 flash stimulus and 20 Hz flicker protocol. Mice were dark-adapted for a 12 hour period and ERG recordings were performed using the Espion ERG Diagnosys system. Pupils were first dilated with drops of both 1% AK-Pentolate (Cyclopentolate Hydrochloride) and 10% AK-Dilate (Phenylephrine Hydrochloride).
• mice were anesthetized with a ketamine (75 mg/kg) and dexmedetomidine (1.0 mg/kg) cocktail (injected i.p.). Celluvisc was placed on the eyes followed by surface electrodes. The ERG was then recorded with the animal on a heating pad (takes 7-8 minutes). Animals were revived by administration of 0.5 mg/kg atipamezole (injected i.p.), returned to their cages, and warmed using a heating pad and monitored until awake.
• Tissue Harvest :
• mice were euthanized with C0 2 , decapitated (to facilitate exsanguination of the periorbital region), eyes excised, and then marked along their superior aspect with tissue marking dye.
• Eyes were immersion fixed over night at room temperature in Davidson's Fix special (BBC Biochemical, Mt Vernon, WA), dehydrated for 1 hr in 50% EtOH at room temperature, and then placed into 70% EtOH at room temperature. Eyes were stored in 70%) EtOH until subjected to automated paraffin processing. After processing, eyes were embedded in paraffin, sectioned on a rotary microtome at 5 ⁇ thickness through the optic nerve head parallel to the vertical meridian, and mounted on glass slides. Sections were baked onto slides, subjected to automated H&E staining, and covers lipped using permanent mounting medium. Stained tissue sections were then examined by an observed blinded to treatment regiments and scored to determine the level of damage to the outer nuclear layer.

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